The major objective of this project is to elucidate the mechanism by which transcription is controlled in eukaryotic cells. Emphasis is placed on the direct role played by multiple DNA-dependent RNA polymerases and on ancillary chromosomal and nonchromosomal proteins which may affect the polymerases or the DNA template. The approach used is to reconstruct an in vitro transcription system for isolated genes (mainly ribosomal RNA genes) and to compare factors affecting that system with changes in in vivo transcription of the same genes during differentiation of a simple protozoan, Acanthamoeba castellanii. Recombinant DNA techniques are being polymerase stimulatory protein have been purified to homogeneity so that a completely isolated chromatin and chromatin reconstituted from purified chromosomal proteins plus recombinant DNA and on chromatin will be made using hybridization, symmetry tests and initiation sequence determinations. RNA polymerases and chromatins isolated from stages in the life cycle of Acanthamoeba which demonstrate active transcription or inactivity of particular genes will be compared.